Die Rheumatoide Arthritis ist eine Autoimmunerkrankung, die mit grundlegenden Veränderungen im CD4+ T-Zellpool einhergeht. Viele Studien konnten zeigen, dass die Gruppe der T-Helferzellen bei erkrankten Personen vermehrt Anzeichen von replikativer Seneszenz und das adaptive Immunsystem deutliche Zeichen der Immunoseneszenz aufweisen. Als zum Teil ursächlich hierfür konnte ein reduzierter Thymusoutput festgestellt werden. Das Ziel der vorliegenden Studie war es, aufbauend auf Erkenntnissen von Six und Kollegen anhand einer Lymphozytenvorläuferzelle, die im peripheren Blut zirkuliert, in vivo in der Lage ist, den Thymus zu besiedeln und sich in reife T-Zellen zu entwickeln, den Thymusinput abzuschätzen, um eine defiziente Thymusbesiedlung als eventuelle Ursache des reduzierten Thymusoutput festzustellen. Hierzu wurde das Blut von 28 Patienten mit Rheumatoider Arthritis und altersentsprechenden gesunden Kontrollen mittels Durchflusszytometrie auf mehrere relevante reife T-Helferzellpopulationen, naive CD4+ T-Zellen und benannte lymphozytäre Vorläuferzelle untersucht. Es konnten dann direkte Vergleiche des prozentualen Anteils bestimmter T-Helferzellgruppen zwischen Erkrankten und Gesunden gezogen sowie Korrelationen verschiedener Zellgruppen untereinander hergestellt werden. Die gewonnenen Ergebnisse wurden mit der hierzu vorliegenden Literatur verglichen und diskutiert.:Abkürzungsverzeichnis Tabellen Abbildungen 1. Einleitung 1.1. Epidemiologie, Ätiologie und Pathogenese der Rheumatoiden Arthritis 1.2. T-Zellen in der Pathogenese der Rheumatoiden Arthritis 1.3. Die T-Zellgenese 1.3.1 Entwicklung im Knochenmark: von der hämatopoetischen Stammzelle zum common lymphoid progenitor (CLP) 1.3.2 Lymphoide Vorläuferzellen in der peripheren Zirkulation 1.3.3 Die Entwicklung von CD4+ und CD8+ einfach positiven T-Zellen im Thymus 1.4. Immunoseneszenz und das periphere CD4+ T-Zellkompartiment bei Rheumatoider Arthritis 2. Material und Methoden 2.1. Patientengut und gesunde Kontrollen 2.2.1. Gewinnung der PBMCs 2.2.2. Gewinnung der PBMCs für das CD31-Experiment 2.3. Immunzytologische Färbung 2.4. Etablierung der Färbeansätze 2.5. Messung am Durchflusszytometer 2.6. Auswertung der Messergebnisse der Durchflusszytometrie 2.6.1 Messungen 1 bis 4 2.6.2. Messung 5 2.6.3. Messung 6 2.7. Erfassung der klinischen Daten der Patienten 2.8. Statistische Auswertung der Ergebnisse 3. Ergebnisse 3.1. Beschreibung des CD4+ T-Zellkompartiments in der peripheren Zirkulation 3.1.1. CD4+CD8+ doppelt positive T-Lymphozyten in der peripheren Zirkulation 3.1.2. Frequenz CD4+ CD28 negativer T-Lymphozyten in der peripheren Zirkulation 3.1.3. Untersuchung der CD85j positiven T-Helferzellpopulation 3.1.4. CD4+CD25+ T-Zellen: aktivierte T-Helferzellen und T-regulatorische Zellen 3.1.5. Quantifizierung von naiven versus Memory-T-Helferzellen 3.2. Recent thymic emigrants: CD31+ thymic naive T-Zellen 3.3. Zirkulierende lymphoide Progenitoren 3.4. CD34 positive hämatopoetische Stammzelle 4. Diskussion 4.1. Das periphere CD4+ T-Zellkompartiment bei der RA 4.2. RA-Patienten weisen eine verringerte Anzahl von RTEs auf 4.3. Die Frequenz von Präthymozyten im peripheren Blut von RA-Patienten ist erhöht 5. Zusammenfassung Literaturverzeichnis Eigenständigkeitserklärung Danksagung

Die Rheumatoide Arthritis (RA) ist eine chronisch-entzündliche Systemerkrankung des Bindegewebes mit autoimmunem Charakter. In dieser Studie wurden 7 Kandidatengene, welche in zentrale Abläufe der Apoptose involviert sind (CFLAR, XIAP, NFKB1, RELA, BCL2L1, FAS, FASLG), selektiert. Innerhalb dieser Gene wurden 23 Einzel-Basen-Polymorphismen (single nucleotide polymorphisms bzw. SNPs) sowie ein Insertions-Deletions-Polymorphismus in 300 französich-kaukasischen Individuen (100 RA-Trio-Familien) mittels Einzelbasenverlängerung (Single Base Extension bzw. SBE) in einer massenspektrometrischen Analyse durch MALDI-TOF-MS (Matrix Assisted Laser Desorption/Ionization–Time Of Flight Mass Spectrometry) genotypisiert. Die Auswahl der zu untersuchenden genetischen Polymorphismen erfolgte dabei unter Berücksichtigung einer möglichen funktionellen Bedeutung, bekannter Assoziationen mit RA oder anderer Autoimmunerkrankungen, der Lage im Gen sowie der genetischen Variabilität. Die Ergebnisse der Genotypisierung wurden genutzt um die Polymorphismen bzw. Kandidatengene mit Hilfe verschiedener statistischer Methoden auf ihre Assoziation mit RA hin zu untersuchen. Die statistischen Analysen des SNPs CFLAR-rs7583529 zeigten hierbei einen nicht signifikanten Trend, wobei das minor Allel A gehäuft in RA Patienten vorkam. Das Ergebnis des Genotypen-Tests (Lathrop) für FAS-rs1800682 belegte einen protektiven Effekt für homozygote Träger des major Allels C (Lathrop pval = 0.045). Unterstützung für die gefundenen Trends bzw. Assoziationen von CFLAR-rs7583529 und FAS-rs1800682 boten Vergleiche mit Daten genomweiter Studien (NARAC/EIRA- und WTCCC-Studie). In den Assoziationsanalysen von BCL2L1-rs3181073 zeigte sich ein protektiver Effekt des minor Allels A (TDT pval = 0.008, OR = 0.51 [0.3 – 0.9], OR pval = 0.014). Der Risikoeffekt des major Allels C spiegelte sich entsprechend im Lathroptest wider, welcher eine signifikante Anreicherung des homozygoten C/C-Genotyps in den Fällen anzeigte (Lathrop pval = 0.021). Die gefundenen Assoziationen von FAS und BCL2L1 mit RA gehen mit der Hypothese konform, dass veränderte Abläufe sowohl im intrinsischen mitochondrialen (BCL2L1) als auch im extrinsischen (FAS) Weg der Apoptose in die Ätiologie der RA involviert sind. Die Ergebnisse dieser Arbeit sollten in einer zweiten unabhängigen Kohorte repliziert werden. In Folgestudien wäre es ebenfalls interessant, weitere SNPs der Kandidatengene zu genotypisieren, um die genetische Variabilität anhand der Haplotypen genauer zu analysieren. Sollten sich die o. g. Assoziationen bestätigen, sind im Weiteren funktionelle Studien bezüglich unterschiedlicher Genexpression oder verändertem Apoptoseverhalten von Zellen oder synovialem Gewebe von großem Interesse. / Rheumatoid arthritis (RA) is a chronic inflammatory systemic disease of the connective tissue with autoimmune character. In this study, 7 candidate genes that are known to be involved in key processes of apoptosis (CFLAR, XIAP, NFKB1, REAL, Bcl2l1, FAS, FASLG) were selected. Within these genes, 23 single nucleotide polymorphisms (SNPs) and one insertion/deletion polymorphism were genotyped in a sample of 300 French Caucasian individuals (100 RA trio families) by means of Single Base Extension (SBE) and MALDI-TOF (Matrix Assisted Laser Desorption /Ionization–Time Of Flight) mass spectrometry analysis. The possible functional significance, known associations with RA or other autoimmune diseases, the location in the gene and genetic variability were taken into account during the selection of genetic polymorphisms. The SNP genotyping results were used to analyse associations of polymorphisms or candidate genes with RA by applying various statistical methods. Analysis of the SNP CFLAR-rs7583529 showed a non-significant trend toward increased frequency of the minor allele A in RA patients. The genotypic test (Lathrop) of FAS-rs1800682 revealed a protective effect for homozygous carriers of major allele C (Lathrop pval = 0.045). Data of genome-wide studies (NARAC/EIRA- and WTCCC study) provided further support for association of CFLAR-rs7583529 and FAS-rs1800682 like confirmed in this study. Association analysis of Bcl2l1-rs3181073 showed a protective effect of the minor allele A (TDT pval = 0.008, OR = 0.51 [0.3 - 0.9], pval OR = 0.014). The genotypic Lathrop-test in turn revealed a corresponding risk effect for homozygous C/C genotype carriers (Lathrop pval = 0.021). Within this study, associations of the apoptosis genes FAS and Bcl2l1 with RA were found out. These results further indicate that changes of the intrinsic mitochondrial (Bcl2l1) and extrinsic (FAS) apoptosis pathway are possibly involved in the etiology of RA. For confirmation, results of this study should be replicated in a larger independent cohort. It would also be of interest to analyze the genetic variability based on specific haplotypes of additional SNPs within candidate genes. If the aforementioned associations are confirmed, functional studies with regard to different gene expression or changed apoptosis initiation in cells or synovial tissue would be of interest.

-:INHALTSVERZEICHNIS I BIBLIOGRAPHISCHE BESCHREIBUNG II REFERAT III ABKÜRZUNGSVERZEICHNIS IV 1 EINLEITUNG 5 1.1 Rheumatoide Arthritis 5 1.2 Stellenwert von Biomarkern bei Rheumatoider Arthritis 5 1.3 Massenspektrometrie 6 1.3.1 Einführung in die Massenspektrometrie 6 1.3.2 MALDI MS Imaging 7 1.3.2.1 Vorteile von MALDI MS Imaging 8 1.3.2.2 Nachteile von MALDI MS Imaging 8 1.3.3 Massenspektrometrie in der Arthritisforschung 9 1.4 Histopathologie bei Rheumatoider Arthritis 9 1.5 Potentielle Biomarker bei Rheumatoider Arthritis 10 1.6 Fragestellung 11 2 PUBLIKATIONSMANUSKRIPT 12 3 ZUSAMMENFASSUNG 17 4 LITERATURVERZEICHNIS 20 5 ANHANG 27 5.1 Selbständigkeitserklärung 27 5.2 Lebenslauf 28 5.3 Danksagungen 30

In dieser Arbeit wurde die Auswirkung von IL-1β sowie der Inhibierung von Il-1β bzw. seines Rezeptors auf die regulatorischen T-Helfer-Zellen (Tregs) gesunder Probandinnen, JIA- und RA-Patientinnen untersucht. Der größte Einfluss von IL-1β zeigte sich bei den untersuchten Zellen der gesunden Probandinnen. Unter IL-1β Stimulation wurde der Treg-spezifische Transkriptionsfaktor FoxP3 signifikant vermindert von den regulatorischen T-Helfer-Zellen, den induzierten regulatorischen T-Helfer Zellen und den Nicht-Tregs der gesunden Probandinnen exprimiert. Ebenfalls zeigte sich ein nicht-signifikanter Trend für eine gesteigerte IL-17 Produktion unter IL-1β Stimulation bei den Tregs der gesunden Probandinnen, der JIA- und der RA-Patientinnen und bei den Nicht-Tregs der gesunden Probandinnen. Dies war bei der IL-1β Inhibierung bzw. der Inhibierung des IL-1 Rezeptors nicht zu beobachten. / In this work, the influence of IL-1β or the inhibition of IL-1β or its receptor on regulatory T helper cells (Tregs) of healthy donors, JIA- or RA-patients was analysed. The biggest effect of IL-1β was observed in cells of healthy donors. After stimulation with IL-1β, expression of the Treg-specific transcription factor FoxP3 was significantly reduced in regulatory T cells, induced regulatory T cells and non-regulatory T cells of healthy donors. Also, a non-significant trend towards increased IL-17 production was found in Tregs of healthy donors, JIA- and RA-patients after IL-1β stimulation. This was not observed after inhibition of IL-1β or the IL-1β receptor.

Autoimmune diseases such as rheumatoid arthritis (RA) are associated with debilitating chronic inflammation, autoantibody production, articular bone erosions and systemic bone loss. The underlying mechanisms and cell types that initiate these diseases are not fully understood, and current therapies mainly address downstream mechanisms and do not fully halt disease progression in all patients. Moreover, previous studies have largely focused on the role of adaptive immunity in driving these diseases, and less attention has been given to the contribution of innate immune pathways such as DNA sensor signaling pathways in initiating and/or perpetuating autoimmunity and erosive inflammatory arthritis. Detection of microbial nucleic acids by DNA sensors such as endosomal toll-like receptors (TLRs) and cytosolic sensors is an early form of antiviral defense. Upon detection of nucleic acid, TLRs dependent on Unc93B and cytosolic sensors dependent on the adaptor stimulator of interferon genes (STING) orchestrate production of type 1 interferons and pro-inflammatory cytokines to resolve infection. Additionally, the cytosolic DNA sensor absent in melanoma 2 (AIM2), which is not dependent on STING, also recognizes microbial DNA and coordinates the cleavage of pro-IL-1β. Previous studies have largely focused on the role of these DNA sensors in macrophages and dendritic cells in the context of antiviral immunity. In recent years, however, the inappropriate recognition of host nucleic acids by these sensors has been associated with several autoimmune diseases including RA. This dissertation aims to delineate the mechanisms by which DNA sensors contribute to inflammatory arthritis and bone remodeling in the context of a murine model of autoimmunity. In DNase II deficient mice, excessive accrual of undegraded, endogenous DNA leads to robust production of type 1 interferons (IFNs) and proinflammatory cytokines. The high levels of type 1 IFNs result in anemia and embryonic lethality; therefore, the gene for the type 1 IFN receptor (IFNaR) has also been deleted so that the mice survive. DNase II-/- IFNaR-/- double knockout (DKO) mice develop erosive inflammatory arthritis, anti-nuclear antibodies, and splenomegaly not seen in the DNase II+/- IFNaR-/- (Het) control group. To evaluate whether cytosolic or endosomal DNA sensors contribute to the clinical manifestations of DKO mice, genes involved in TLR or cytosolic sensor signaling were deleted on the DKO background. Genetically altered mice include STING/DNaseII/IFNaR TKO (STING TKO), AIM2/DNase II/IFNaR TKO (AIM2 TKO), and Unc93b/DNase II/IFNaR TKO (Unc93 TKO) mice. Our hypothesis was that the STING, AIM2, and/or Unc93 pathways would contribute to the autoimmune manifestations in DNase II deficient mice. Rigorous examination of inflammation in these lines revealed important roles for both the STING and AIM2 pathways in arthritis. Despite the substantial effects of the STING and AIM2 pathways on arthritis, STING TKO and AIM2 TKO mice still exhibited prominent autoantibody production. Interestingly, inflammation persisted in Unc93 TKO mice while autoantibody production to nucleic acids was abrogated. Collectively, these data indicate that innate immune pathways contribute to the initiation/perpetuation of inflammatory arthritis and demonstrate that cytosolic and endosomal pathways play distinct roles in the manifestations of autoimmunity. Moreover, they reveal a previously undescribed role for AIM2 as a sensor of endogenous nucleic acids in inflammatory arthritis. Thus, therapeutics that target the STING and AIM2 pathways may be beneficial for the treatment of inflammatory joint diseases. While the role of hematopoietic cells in driving autoimmunity has been well established, the contribution of stromal elements to disease pathogenesis is less well understood. Therefore, we generated bone marrow chimeras to delineate the contribution of hematopoietic and non-hematopoietic cells to the various autoimmune manifestations in DKO mice. These studies revealed that both donor hematopoietic and host radioresistant cells are required for inflammation in the joint as well as for other features of autoimmunity in DKO mice, including splenomegaly, extramedullary hematopoiesis, and autoantibody production. This data demonstrates that stromal host cells play a major role in DNA-driven autoimmunity. Moreover, these results suggest that targeting not only hematopoietic but also stromal elements may be advantageous in the setting of inflammatory arthritis. In the final chapter of this thesis, a role for innate immune sensor pathways in bone is described. The majority of inflammatory arthritides have been shown to lead to systemic loss of bone. Surprisingly, however, we found that DKO mice accumulate trabecular bone in the long bones over time as well as ectopic bone in the spleens, both sites of robust DNA accrual. Moreover, deficiency of the STING pathway abrogated this bone accumulation. Collectively, these data demonstrate that DNA accrual promotes dysregulated bone remodeling through innate immune sensing pathways. These findings are the first to reveal a role for the STING pathway in bone and may unveil novel targets for the treatment of diseases associated with bone disorders.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20971号 / 医博第4317号 / 新制||医||1026(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 河本 宏, 教授 濵﨑 洋子, 教授 松田 秀一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

A psychological intervention involving relaxation training and biofeedback training for the control of peripheral skin temperature was investigated in this study with 27 female rheumatoid arthritics as participants. Based on analysis of the temperature data, it was concluded that the biofeedback response was not learned. From electromyographic data, it was concluded that participants did learn to relax. The hypothesis that the two treatment components would have beneficial effects on the physical, functional, and psychological aspects of rheumatoid arthritis was answered partially. No differential effects as a function of biofeedback training were found as the data for the temperature increase and temperature decrease groups were statistically combined in multiple analyses of variance for repeated measures. Although no differential effects were obtained, numerous positive changes were found. Correlated with the relaxation training were decreases in reported subjective units of discomfort, percentage of time hurting, percentage of body hurting, and general severity of pain. Improved sleep patterns were reported as was increased performance of activities of daily living. Reductions were also found in psychological tension, and in the amount of time mood was influenced by the disease. Shifts were not found in imagery, locus of control, and other psychological dimensions. Constitutional improvements were also absent.

No description available.

Introduction Disease monitoring in rheumatoid arthritis (RA) can be improved by incorporating imaging technologies. Clinical examination fails to detect subclinical inflammation in half of metatarsophalangeal joints (MTPJs), but the effectiveness of using ultrasonography (US) in MTPJs is unclear. We aimed to evaluate US assessment of disease activity in the MTPJs using MRI as the reference standard, in comparison to clinical examination. Methods Patients newly diagnosed with RA (ACR criteria) were recruited and assessed at baseline, 6 weeks, 3 months, 6 months, and 12 months. A rheumatologist assessed the MTPJs 2-5 bilaterally for swelling and tenderness (presence=1), and for erosion (presence=1), synovial thickening, and power Doppler (PD) by US. Synovial thickening and PD were graded semi-quantitatively (grade 0-3). The most clinically symptomatic foot was scanned using extremity MRI (1.0T) at the baseline and 12-month visits. MTPJs 2-5 were graded semi-quantitatively for synovitis, bone marrow edema (BME) (grade 0-3), and erosions (grade 0-10). Results Forty-one patients were recruited (mean (SD) age=51.9 (10.3) years, 81% female). Kappa agreement was moderate between PD and grade ≥2 synovitis (k=0.46) and BME (k=0.47), but poor agreement was found for clinical examination and synovial thickening. US was able to visualize subclinical inflammation in 41% of non-swollen joints. After 12 months, the average total score for synovial thickening, PD, and BME all significantly decreased, but not swollen or tender joint counts. US visualized few erosions (n=8) compared to MRI (n=101) in the most symptomatic foot. MRI observed erosion repairs in patients treated with DMARDs, and repairs appeared to be preferential for MTPJs that had low inflammation seen by US. Conclusion US appears to better visualize MTPJ inflammation than swollen and tender joint counts, and may be used in combination with clinical examination to improve routine disease monitoring in RA. / Thesis / Master of Science (MSc) / Current assessment of rheumatoid arthritis (RA) relies on physical examination for joint swelling and tenderness, but these methods often miss underlying inflammation. Ultrasonography (US) may help improve the diagnosis and monitoring of disease activity, but its effectiveness in imaging the feet is unclear. With MRI (another imaging technology) as the standard of reference, we compared the use of physical examination and US in assessing inflammation in the feet, and explored the potential of using US to see damage to bones. In 41 patients with early RA (<2 years of disease), US was able to detect inflammation in many joints that appeared normal, and was better at detecting decreased inflammation over 1 year than physical examination. Although US was limited at assessing early bone damage, it was able to see several large erosions. In conclusion, US can better visualize disease activity than clinical examination and can improve RA assessments.